The subject of this proposal is cholesterol metabolism, in particular, the regulation of the key enzyme, hydroxymethyl glutaryl coenzyme A reductase. The questions we intend to answer concern the biochemical properties of the enzyme and the mechanisms regulating its activity. Information is essential to an understanding of the physiological alterations in cholesterol metabolism due to dietary intake of cholesterol and due to various abnormal conditions. Techniques for purifying the enzyme have been developed in the past two years and some knowledge of its characteristics has been obtained. The observation of reversible cold inactivation and the concomitant alteration of subunit structure of the soluble reductase is an extremely important lead. A reversible alteration of the physical state is a potential regulatory mechanism and will be investigated. Purification of the enzyme to homogeniety is essential to our understanding of its structure and will allow preparation of specific antibody with which to monitor physiological alterations in enzyme levels. We propose to continue a detailed examination of the role of the endoplasmic reticulum membrane itself on the regulation of enzyme activity. Preliminary observations suggest that the temperature dependent alteration in the catalytic properties of the soluble enzyme are quite different from those of the in situ microsomal enzyme suggesting that changes in the structure of the microsomal membrane could affect catalysis.